Certain microorganisms such as bacteria, fungi and yeasts are known to convert organic molecules to lipids. Oil produced by heterotrophic microorganisms is often called as single cell oil or microbial oil and lipid producing microorganisms are called oleaginous microbes.
Process for production of single cell oil typically comprises steps of cultivating microorganisms, allowing cells to accumulate lipids, harvesting lipid-rich cells and recovering lipid from microbial cells. Microorganism-based lipids (i.e. single cell oils) can be used as raw materials for production of biofuels such as biodiesel, renewable diesel or bio jet fuel.
The oleaginous microorganisms cultivated on a growth medium can contain lipids up to 80% of their total dry matter content. The efficiency of the method used to recover lipids from microbial biomass is crucial to the economically feasible production of biofuels by microbial fermentation. The method used for lipid recovery should provide around 95% oil yield in order to be economically feasible. Furthermore, the method used to recover the lipids should not destroy the value of the residual biomass, from which oil has been extracted or to decrease the quality of the extracted oil, which can be further processed or used as such.
A lipid recovery process typically comprises steps of harvesting the microbial cells from cultivation medium, disrupting the microbial cells and recovery of oil to obtain crude microbial oil and a residual biomass fraction. The oil can be recovered from wet biomass or in case of dry extraction, the microbial biomass is dewatered before oil recovery. The residual biomass fraction may be used as animal feed since it contains valuable protein fractions or used for energy production through combustion. The method to recover lipids basically determines the value of the residual biomass as animal feed since the proteins of microbial biomass are easily degraded during the various process steps.
The harvesting step is used to separate microbial cells from cultivation medium. Conventional harvesting techniques include filtration and centrifugation. The microbial biomass can also be concentrated before drying step by mechanical dewatering means before the recovery of lipids.
Oil is usually recovered from the harvested microbial cells by extraction with a solvent such as carbon dioxide in subcritical or supercritical state or liquid hydrocarbons such as hexane. Microbial cells may also be disrupted before extraction of lipids with methods such as ultra-sonication, osmotic shock, mechanical shear force such as extrusion, cold press, and thermal shock. The need to completely disrupt the microbial cells depends on the solvent used to extract lipids. In case complete disruption of cells is not possible, a mixture of non-polar and polar solvents can be used in extraction.
In dry extraction the harvested biomass is dried before solvent extraction to remove as much free water as possible. The lipids can also be extracted from wet biomass. In the dry extraction the microbial biomass is typically dried to a dry matter content of above 90% before solvent extraction. In wet route extraction the dry matter content of the biomass can be as low as 5% in the solvent extraction step. In case of wet extraction the resulting wet residual biomass fraction needs to be dried if it is used in value added applications.
Traditional cell harvesting techniques such as filtration and centrifugation usually result in a biomass with dry matter contents from 15% up to 40%. The dry matter content of harvested biomass can be further increased by drying, which, however, consumes a lot of energy. Drying can be performed by heating, freeze drying or spray drying (Farr, W. E. and Proctor, A., Green Vegetable Oil Processing, 2012, AOCS Press). Generally biomasses having dry matter contents of below 30 w-% are not considered economical to dry.
The selection between dry and wet route extraction depends on the ease of removal of excess liquid from microbial biomass. Wet route extraction is usually preferred if biomass cannot be dewatered mechanically to above 30% dry mater content. Wet extraction is also preferred if the subsequent processing of the residual biomass does not require further drying, e.g. if biomass is to be processed by anaerobic digestion or by hydrothermal liquefaction.
The ease of removal of water from wet microbial biomass depends on the type of microorganisms in the microbial biomass. Filamentous organisms are considered much easier to harvest due to their large cell size whereas algal biomass is known to have poor filterability due to the small cell size. Wet route extraction has been suggested as an efficient lipid recovery method for algal biomass in EP2450424A1 as it is clearly stated that it is not necessary to dry the wet microbial biomass before extraction. Thus EP2450424A1 does not relate to the filtration and drying steps before extraction. Moreover, the document refers to an elevated pressure of the extraction itself since propane is used which requires a pressure of at least 20 bar during the extraction step in order to keep the extractant/solvent in liquid state. With respect to the heating step, there is no mentioning of the exact pressures used. The examples according to the document reveals rather low yields of lipids using the method as disclosed in the document and there is no mentioning of the content of nitrogen in the extracted oils from the biomass.
Water present in the harvested cells is known to decrease the efficiency of the solvent extraction since it shields the hydrophobic lipids from solvent contact. Additionally, water tends to form emulsions with the solvent, which furthermore decreases the extraction efficiency and makes it more difficult to separate extracted lipids from the rest of the liquid phase. Consequently, the dry extraction is usually preferred over wet extraction if the microbial biomass can be mechanically dewatered to above 30% dry matter content.
It is has been previously disclosed by Davies (Davies, R. 1992. Scale Up of Yeast Oil Technology. Industrial Applications of Single Cell Oils. Edited by: D. J. Kyle and C. Ratledge, AOCS Publishing) that a nozzle disc separator, which is typically used in the brewing and baker's yeast industry to harvest yeast, is not efficient for harvesting oleaginous yeast when the oil content of the biomass is over 35% on a dry weight basis. With oleaginous yeast biomasses that have higher oil content than 35% on a dry weight basis, cross flow filtration has been suggested. However, this harvesting method has the disadvantage of low flux and membrane fouling, which mean that large membrane areas are needed.
Even if nozzle disc separator is used to harvest oleaginous yeast with oil content under 35% on a dry weight basis, the resulting concentrated yeast cream has a dry matter content of 15-20%. This is universally deemed too low dry matter content for the drying to be economical. For this reason, the wet extraction is commonly preferred with oleaginous yeast with oil content lower than 35% on a dry weight basis harvested using a nozzle disc separator.
WO2011/143380 discloses a method for separation of oil from algal cells, in which method wet algal biomass is subjected to hydrothermal carbonization treatment (HTC), the resulting combined oil and char fraction are separated from aqueous fraction by filtration, and the oil is separated from char fraction by solvent extraction. The hydrothermal carbonization treatment is conducted at a temperature of from 170 to 225° C. The method has the advantage that it produces an aqueous fraction rich in nitrogen, phosphorus, and potassium, which can be reused in the growth stage as nutrients. The hydrothermal carbonization treatment is not reported to have any effect on filterability of the biomass or to efficiency of the extraction step. However, the document appears to exemplify methods based on process distiller grains and other raw materials. This type of raw material does not contain much lipids and as such would not present any difficulties in filtering the wet biomass where the method does not include a thermal treatment step prior to filtration. Moreover, in order to achieve high yield of the fatty acid methyl esters (FAMEs) it is necessary to both extract the dry char as well as the aqueous filtrate. Moreover, the reference is silent with respect to the quality of the retrieved oils with respect to e.g. nitrogen or phosphorus contents. In fact, the document clearly states that the aqueous product contains most of the nitrogen, phosphorus and potassium originally present in the biomass and extracting the aqueous phase would dearly result in a nitrogen content in level with the nitrogen content of the original biomass.
Microbial biomass is also commonly pasteurized in the harvesting step in order to kill the microorganisms and deactivate enzymes, which might otherwise destroy the lipid structures. In the method disclosed in US 2003/0143659, the pasteurization is conducted by heating the biomass in its growth medium to a temperature of 60-100° C. for up to 90 minutes. The pasteurization was not reported to have any effect on subsequent dewatering or lipid extraction steps.
EP2450426 relates to a purification method for purifying lipid materials already obtained from biological material. The method comprises the use of at least one polar solvent and at least one non-polar solvent and demonstrates how various reaction conditions influence the contents of phosphorus and metals in the purified oils. However, the document is silent with respect to the nitrogen content of the extracted oils and is also silent with respect to any methods of extracting oils from a biomass or how any conditions during extraction may influence the contents of e.g. nitrogen in the extracted oils.
The present invention is related to recovery of lipids from oleaginous yeast biomass and to a method for producing lipids using the method for recovery of lipids.
Davies (Davies, R. 1992. Scale Up of Yeast Oil Technology. Industrial Applications of Single Cell Oils. Edited by: D. J. Kyle and C. Ratledge, AOCS Publishing) reports poor recovery of oleaginous yeast cells, when the lipid content of the biomass exceeds 35% on a dry weight basis.
Harvesting of oleaginous yeast cells with dead end micro filters was found to be impossible due to the immediate blinding of filter membranes due to the soft cell structure of yeast biomass. The experiments also indicated that it was possible to harvest oleaginous yeast cells with a cross flow filtration arrangement but the filtration result was poor with only 28% dry matter content of harvested biomass, which is generally considered too low for the subsequent drying step. It was also found out that the presence of lignocellulosic material or remains of lignocellulosic material in the biomass suspension to be harvested makes the fouling of the membranes even worse.
Consequently, there is need for an effective method for recovery of lipids from oleaginous yeast biomass and especially from yeast biomasses having high lipid content, i.e. over 35% on a dry weight basis, such as e.g. at least about 40% on a dry weight basis, or such as e.g. at least about 50% on a dry weight basis, such as e.g. at least about 60% on a dry weight basis, such as e.g. at least about 70% on a dry weight basis, such as e.g. at least about 80% on a dry weight basis